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UNIVERSITI PUTRA MALAYSIA

MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF TWO VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS

ISOLATES, UPM94/273 AND UPM97/61

KONG LlH LING

FPV 2003 2

MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF TWO VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS ISOLATES,

UPM94/273 AND UPM97/61

By

KONG LlH LING

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for

the Degree of Master of Science

February 2003

Dedicated with love and gratitude to:

My dearest parents, fiance, family and five lovely nieces

11

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF TWO VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS ISOLATES,

UPM94/273 AND UPM97/61

By

KONG L1H LING

February 2003

Chairman : Associate Professor Dr. Abdul Rahman Omar, Ph.D.

Faculty: Veterinary Medicine

An atypical very virulent (w) strain (UPM94/273) and typical vv strain

(UPM97/61) of infectious bursal disease virus (IBOV) isolated in

Malaysia, were characterized both in vivo and at the molecular level.

Comparison of the deduced amino acid sequences with other serotype 1

and 2 sequences revealed 16 amino acid residues, which were

conserved only in the vvIBDV. Among the 16 unique amino acid

differences, 8 were in VP1 (146 Asp, 147 Asn, 242 Glu, 390 Met, 393

Asp, 562 Pro, 687 Pro and 695 Arg), 3 were in VP2 (222 Ala, 256 lie and

294 lie), 2 were in VP3 (990 Val and 1005 Ala) and 3 were in VP4 (685

Asn, 715 Ser and 751 Asp). The importance of these unique amino acid

residues is not known but they could affect the virulence of vvIBOV. The

UPM94/273 also demonstrated 6 unique amino acid residues at segment

111

A at positions Ser254, Glu270, Lys588, Ser745, Phe838 and Lys863 and

8 unique amino acid residues at segment B at positions Ala92, Ser1 00,

Va1208, Asp253, Asp560, Asn565, Gly750 and Gly876. In addition, these

amino acid substitutions have not been reported before in wlBDV and

were found only on variant, classical and/or serotype 2 strains. However,

the VP5 region of both wlBDV strains was conserved. The

UPM97/61 demonstrated 7 unique amino acid substitutions at segment A

and 4 unique amino acid substitutions at segment B . However, none of

the amino acids changes have been reported elsewhere in other IBDV

strains. Although the actual functions of the amino acid substitutions are

not know, the unusual amino acid substitutions at segment A and/or B of

both isolates may be important in virus viru lence. Alignments of the

nucleic acid and amino acid sequences of segment A and B followed by

distance analysis al lowed the generation of phylogenetic trees.

Phylogenetic analysis based on segment A and B revealed that all the

wlBDV strains including UPM94/273 isolate can be clustered in a group

that is d istinct from classical , variant, attenuated and serotype 2 strains.

However, the tree branching patterns were qu ite different between

segment A and segment B. in addition , the wlBDV strains showed

several conserved amino acid substitutions at segment B as found in the

Australian 002-73 and serotype 2 strains. These findings indicate that

probably a genetic reassortment may have play an important role in the

emergence of vvIBDV. Flow cytometry and real time peR assays,

indicated that chickens infected with UPM97/61 induced higher

IV

percentages of apoptotic cells but lower level of viral load whereas

UPM94/273 induced lower percentages of apoptotic cel ls but higher level

of viral load, suggesting a negative correlation between viral load and

apoptosis. These results indicated that UPM97/61 was more virulent than

UPM94/273.

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENCIRIAN MOLEKUL DAN BIOLOGIK ISOLATISOLAT UPM94/273 DAN UPM97/61 VIRUS PENYAKIT BURSA BERJANGKIT

Oleh

KONG L1H LING

Februari 2003

Pengerusi: Profesor Madya Dr. Abdul Rahman Omar, Ph.D.

Fakulti : Perubatan Veterinar

Virus penyakit bursa berjangkit amat virulen (wi OBV) yang tidak khusus

(UPM94/273) dan yang khusus (UPM97/61 ) d iperolehi dari Malaysia,

telah d icirikan dalam vivo dan berdasarkan molekul . Perbandingan

jujukan asid amino dengan jujukan serotip 1 dan 2 yang lain telah

menunjukkan 1 6 asid amino residu yang cuma terdapat d i wIBOV.

Perbezaan antara 1 6 asid amino yang unik ini , 8 terdapat di VP1 ( 146

Asp, 1 47 Asn, 242 Glu, 390 Met, 393 Asp, 562 Pro, 687 Pro dan 695

Arg), 3 terdapat di VP2 (222 Ala, 256 lie dan 294 l ie), 2 terdapat di VP3

(990 Val dan 1 005 Ala) dan 3 terdapat di VP4 (685 Asn, 71 5 Ser dan 751

Asp). Kepentingan residu-residu asid amino ini adalah tidak diketahui

tetapi ia boleh mempengaruhi viru len vvIBOV. UPM94/273 juga

menunjukkan 6 asid amino yang unik pad a kedudukan Ser254, Glu270,

VI

Lys588, Ser745, Phe838 dan Lys863 mana setiap 2 berada d i VP2, VP3

dan VP4. Tambahan pula, penggantian asid amino pad a kedudukan

Gly254Ser, Ala270Glu, Glu588Lys dan Asp745Ser cuma terdapat di

varian , klasik dan/atau serotip 2. Walaubagaimanapun, bahagian VP5

untuk kedua-dua wlBOV adalah kekal. UPM97/61 menunjukkan 7

penggantian asid amino yang unik d i segmen A dan 4 penggantian asid

amino yang unik d i segmen B. Tetapi, perubahan asid amino in i tidak

pernah dilaporkan pada strain-strain IBOV yang lain. Walaupun fungsi

sebenar penggantian asid amino adalah tidak diketahui, tetapi

penggantian asid amino yang luar biasa ini pada segmen A dan/atau B

untuk kedua-dua isolat mungkin penting dalam virulen virus. Susunan

untuk jujukan asid nukleik dan asid amino untuk segmen A dan B diikuti

oleh anal isis jarak membenarkan pembentukan pokok filogenetik.

Analisis filogenetik berdasarkan segmen A dan B menunjukkan semua

strain wlBOV termasuk isolat UPM94/273 IBOV boleh d ikumpulkan

dalam satu kumpulan d i mana adalah berbeza daripada strain-strain

klasik, varian, akenuat dan serotip 2. Walaupun begitu , corak-corak untuk

cabangan pokok adalah berlainan antara segmen A dan segmen B.

Tambahan pula, strain-strain wlBOV menunjukkan beberapa

penggantian asid amino yang kekal dalam segmen B seperti yang

dijumpai dalam strain-strain Australian 002-73 dan serotip 2. Penemuan

ini menyatakan bahawa kemungkinan penyusunan semula genetic yang

mungkin memainkan peranan yang penting dalam kemunculan wIBOV.

Cara-cara aliran sitometri dan PCR masa sebenar, menyatakan bahawa

VB

ayam-ayam yang dijangkiti dengan UPM97/61 mengakibatkan peratusan

sel-sel apoptosis yang lebih tinggi tetapi takat muatan virus yang lebih

rendah manakala UPM94/273 mengakibatkan peratusan sel-sel

apoptosis yang lebih rendah tetapi takat muatan virus yang lebih tinggi,

mencadangkan satu perhubungan yang negatif wujud d i antara muatan

virus dan apoptosis. Keputusan ini menyatakan bahawa UPM97/61

adalah lebih virulen daripada UPM94/273.

Vlll

ACKNOWLEDGEMENTS

I wou ld l ike to express my heartiest gratitude and appreciation to

Dr. Abdul Rahman Omar, chairman of the supervisory committee for

providing invaluable advice and untiring assistance in this master

research. His constant guidance, participation in laboratory works and

mostly for sharing knowledge on molecular technique that enabled me to

accomplish my work. Moreover, I would l ike to express my sincere thanks

and appreciation to Professor Dr. Aini Ideris, Associate Professor Dr.

Mohd Hair Bejo, Associate Professor Dr. Seow Heng Fong , members of

the supervisory committee for their constructive suggestion, proper

guidance and encouragement throughout my study period .

I am grateful to Maha Abdullah for helping and guiding me in

handling flow cytometry technique; guidance from Dino and Gabrielle in

real time peR approach, and also Siaw Liung and Tzer Miin for their

helpful guidance in SPSS statistical analysis. Special thanks also go to

the staffs in Biologics Lab, Puan Rodiah Husin and Mr. Mohd Redha

Izwan B. Adnani ; and friends Sheau Wei , Siti, Shila, Ain i , Balkis, Lee

Kim, Su Fun, May Ling, Keng Fei, Zul , Thapa, Do Yew, Sok Fang and

every body for always being so willing to render assistance throughout

the course of my study.

IX

I certify that an Examination Committee met on 25th February 2003 to conduct the final examination of Kong Lih Ling on her Master of Science thesis entitled "Molecular and Biological Characterization of Two Very Virulent Infectiuos Bursal Disease Virus Isolates, UPM94/273 and UPM97/61" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the candidate be

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